Microfilaments are ubiquitous structures of all eucaryotic cells which, through their associations with the cytoplasmic membrane, perform both a cytoskeleton role and are involved in many forms of cell motility. Different microfilament systems exist, yet little is known about their protein compositions or how they attach to membranes. The intestinal brush border provides a model system for study. It contains three morphologically distinct systems of microfilaments. In addition, there are both end-on and lateral attachment of microfilaments to the microvillus membrane. Two complementary projects are proposed. The first involves a detailed biochemical and ultrastructural analysis of the proteins involved in microfilament-membrane attachments within the microvillus. Methods developed in this lab have allowed the isolation of intact microvilli from brush borders. They retain their microfilament core, their microfilament-membrane attachment at the tip, and the regular array of cross-filaments which link the core laterally to the membrane. It is proposed to isolate and raise antibodies to the microfilament-membrane attachment proteins and localize them in the microvilli by immuno-electron microscopy. Biochemical analyses will be used to elucidate the disposition of these proteins with respect to the microvillus membrane and to study their interaction with F-actin. The second project involves the identification, purification, and localization of the protein components associated with the different microfilament systems of the brush border. These proteins will be isolated, antibodies raised, and used in immuno-electron microscopy to localize them in the brush border.